Egyptian Journal of Pathology:
Predictive value of immunohistochemical markers of invasive breast cancer, such as estrogen, progesterone receptors, HER-2, and CK5/6, in young Egyptian women with BRCA1 mutation
El-Naggar, Samia I.a; Daoud, Sahar A.a; Abu Gabal, Khadiga M.b; Kamal, Manalc; Shaaban, Yasser Hassan M.c; Shehata, Mohamedc
Departments of aPathology
bClinical and Chemical, Faculty of Medicine, Beni-Suef University, Beni-Suef
cClinical and Chemical Department, Faculty of Medicine, Cairo University, Cairo, Egypt
Correspondence to Samia I. El-Naggar, MD, Department of Pathology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt e-mail: firstname.lastname@example.org
Received February 24, 2013
Accepted March 9, 2013
Background: Breast cancer is a major cause of cancer-related death in women and is increasing in incidence. Although this tumor generally appears in sporadic form, between 5 and 10% of cases can be considered hereditary, attributable to inherited mutations in several susceptibility genes. However, evidence on the effect of breast cancer susceptibility gene 1 (BRCA1) or BRCA2 mutation on prognosis is inconsistent.
Materials and methods: Sixty Egyptian women under the age of 40 years were studied. Archival formalin-fixed paraffin-embedded breast cancer tissues were obtained from pathology files. The histopathology and grade of the tumor were assessed. Immunohistochemical study and RNA extraction were also performed. Cells from the MCF-7 cell line grown in Roswell medium and known to express BRCA1 were processed in paraffin and used as positive controls for both immunohistochemical analysis and real-time reverse transcription-PCR. A study was performed to compare the expression of prognostically meaningful immunohistochemical markers such as estrogen receptors, progesterone receptor, HER-2, and CK5/6 in tumor cells of young female patients with breast cancer with or without mutation in the BRCA1 gene.
Results: Alteration in BRCA1 mRNA expression was found in 40% of cases. A positive relationship was demonstrated between lack of BRCA1 expression and high histological grade, negative estrogen and progesterone receptor status, and overexpression of HER-2 in breast cancer tissues.
Conclusion: The study demonstrated an inverse relationship between BRCA1 expression and parameters that determine poor prognosis in breast cancer.
Breast cancer is a major cause of cancer-related mortality in women (Hill et al., 1997). To some extent, the high proportion of cases in young women reflects the high prevalence of hereditary breast cancer (Parkin et al., 2000). An accurate prognosis is necessary not only to determine the natural history of the disease but also to establish the appropriate treatment method. A better prognostic stratification of patients is important so that patients can receive treatment at an earlier stage and unnecessary risk to those patients who do not need additional treatment can be avoided. Several prognostic factors that provide information about either survival or risk of recurrence are available for breast cancer – namely, tumor size, histological type and grade, and hormone receptor analysis. Breast cancer susceptibility gene 1 (BRCA1) and BRCA2 are considered to be ‘gatekeepers’: they are genes whose mutation or altered expression relieves normal controls on cell division, death, or lifespan, promoting the outgrowth of cancer cells. The function of BRCA1 and BRCA2 genes, classified as tumor suppressors, is linked to key metabolic processes such as DNA-damage repair, regulation of gene expression, and cell cycle control (Guerra et al., 2003).
BRCA1-associated breast cancers often occur in younger women; such tumors are of high grade and lack estrogen receptors (ERs) (Jóhannsson et al., 1997; Karp et al., 1997). All these features are associated with poor prognosis. However, evidence on the effect of a BRCA1 or BRCA2 mutation on prognosis is inconsistent (El-Tamer et al., 2004; Brekelmans et al., 2006). Although this tumor generally appears in sporadic form, between 5 and 10% of cases can be considered hereditary, attributable to inherited mutations in several susceptibility genes (Lynch et al., 1994; Hill et al., 1997). It is found that about 40–50% of hereditary breast cancers and most hereditary breast–ovarian syndromes are thought to be due to mutations in the BRCA1 (Easton et al., 1995). The BRCA1 gene, identified by positional cloning in 1994, consists of 24 exons, 22 of which encode for a protein of 220 kDa consisting of 1863 amino acids (Miki et al., 1994). BRCA1 mRNA is induced at late G1/early S phase before DNA synthesis (Vaughn et al., 1996), and the expression of the BRCA1 protein closely follows that of its mRNA (Chen et al., 1996). It has also been reported that ER-negative breast cancer is a type of tumor with poor prognosis and had treatment strategies comparable to those of ER-positive tumors. Recent studies have also shown that BRCA1-related breast cancers tend to have clinicopathological features that are usually associated with a poor prognosis, such as high grade, ER-negative and progesterone receptor (PR)-negative status, and overexpression of HER-2 (Marcus et al., 1996; Yoshikawa et al., 1999; Putti et al., 2005).
Currently, the genetic basis of breast cancer in Egypt is unknown. This work studied the expression of BRCA1 (mRNA) in breast cancers in Egypt using prognostic parameters such as histopathological type and tumor grade, hormone receptor status, HER-2, and CK5/6.
Materials and methods
Sixty patients below 40 years of age (mean age 36 years; range 18–40 years) affected by breast cancer were studied. Clinicoepidemiological data were obtained from the patients’ records from Beni-Suef University and Cairo University Hospitals, Egypt, for the study. Paraffin blocks were prepared; sections of 5 μm thickness were cut from each tissue. Each section was stained with hematoxylin and eosin. Cases of intraduct carcinoma without invasion were excluded. Tumors were graded according to the system of Scarff–Bloom–Richardson (Dalton et al., 1994) into grade I (well differentiated), grade II (moderately differentiated), or grade III (poorly differentiated).
The remaining sections were stored for use in immunohistochemical analysis and RNA extraction. Cells from the MCF-7 cell line grown in Roswell medium (Gibco BRL) and known to express BRCA1 were processed in paraffin and used as positive controls for real-time reverse transcription-PCR (RT-PCR) (Scully et al., 1996). Consent is unavailable as the materials of this study were archival paraffin blocks taken by code number (patients’ identities were unavailable).
Study approved by ethical committee.
Sections of 5 μm thickness were deparaffinized, rehydrated, and treated with 3% H2O2 in methanol for 30 min to block endogenous peroxidase. Epitope retrieval was carried out in 0.01 mol/l citrate buffer at 95°C in a water bath for 20 min. Nonspecific immunostaining was eliminated using rabbit antimouse serum before monoclonal antibodies for 30 min. After overnight incubation at 4°C with the primary antibody in a humidified chamber, biotinylated antibody (link antibody) was added, followed by streptavidin (dilution 1/500). Diaminobenzene was used as chromogen. Interpretation of the immunostaining was carried out according to the usual criteria by two pathologists. ER and PR were considered positive if 10% of tumor cell nuclei with or without cytoplasmic staining were immunoreactive. HER-2 was scored positive if at least 50% of tumor cell membranes were immunostained. Cytoplasmic immunoreaction for CK5/6 was also studied. Sections were treated in a similar way except that they were incubated in primary antibody for 90 min at the appropriate dilution [monoclonal (ID5) ER 1/50, polyclonal PR 1/50; Dako A/S, Denmark].
RNA extraction and real-time RT-PCR
The rehydrated sections were scraped off the glass slide into an Eppendorf tube containing RNase inhibitor, proteinase K buffer (pH=8.3), 1% Tween surfactant, and proteinase K (10 mg/ml). The tubes were kept overnight (16 h) at 60°C in a water bath and then mixed with 400 μl Trizol and chloroform to precipitate the RNA. To control for RNA degradation, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA had to be amplified in RNA samples extracted from breast tissue sections before BRCA1 expression in that tissue was deemed negative. There was no need to treat the samples with DNase because BRCA1 probe and GAPDH primers were spanning exon/exon junctions (Table 1). RNA was extracted from MCF-7 cells and used as a positive control for RT-PCR. RT-PCR master mixes were prepared according to ThermoScript RT-PCR System (Invitrogen) protocols. Samples for no-RT and no-template were also included in each test to detect any DNA interference or RNA contamination. The ABI7000 real-time machine (Step-One Software; Applied Biosystems, California, USA) was used. Amplification conditions were 60°C for 60 min and 95°C for 5 min, and 45 PCR cycles at 95°C for 15 s and 60°C for 1 min.
Data were collected, coded, and analyzed using SPSS software version 15 (SPSS Inc., Chicago, Illinois, USA) for Windows XP. The following statistical tests were used: the Student t-test and the Mann–Whitney test for not normally distributed data. We investigated the correlation between two parameters using Pearson’s correlation. Significance level was considered at 0.05.
Of the 60 studied cases in the present study, 44 were infiltrating ductal carcinoma, which accounted for 73.3% of the cases graded as grade I (three cases), grade II (24 cases), and grade III (17 cases). Seven cases were infiltrating lobular carcinoma (11.7%) and five cases were mixed ductal lobular carcinoma (8.3%). The remaining four cases were medullary carcinoma, which accounted for 6.7% of the cases. In the 60 studied cases, BRCA1 alteration was seen in 24 cases (40%) and was mostly detected in high-grade invasive ductal carcinoma and medullary carcinoma (17 cases of infiltrating ductal carcinoma grade III, four cases of medullary carcinoma, and three cases of grade II infiltrating ductal carcinoma). The results are summarized in Table 2. The remaining 36 cases showed no alteration in BRCA1 mRNA (24 cases of grade I and II infiltrating ductal carcinoma, seven cases of lobular carcinoma, and five cases of infiltrating mixed ductal lobular carcinoma). There was a significant association between BRCA1 mRNA expression and histological grade of breast cancer, as 21 of 24 cases (87%) were high-grade carcinoma (Fig. 1).
ER, PR, HER-2, and CK5/6 expression in breast cancer tissue
In patients with altered BRCA1 mRNA expression, ER nuclear expression was negative in 66.6% (16 cases), whereas PR nuclear expression was negative in 45.8% (11 cases). HER-2 was overexpressed in eight cases (33% of the cases) (Fig. 2a) with mutation in BRCA1 mRNA (24 cases), one of which was CK5/6 positive (Fig. 2b), although these associations were statistically significant (P<0.05).
Molecular and immunohistochemical techniques provide insights that will allow us to tailor the management of patients with breast cancer. BRCA1 is becoming an important prognostic factor for breast cancer. The morphological features of tumors from patients with breast cancer associated with BRCA1 mutation include higher grade, with an excess of medullary carcinoma (Marcus et al., 1996). Tumors associated with BRCA1 mutation are more likely to be steroid hormone receptor (ER and PR) negative (Osin et al., 1998). The present study showed an alteration in BRCA1 mRNA expression in 40% of tissues studied, with a statistically significant relationship between BRCA1 mRNA expression and poor prognosis as well as high-tumor grade (P<0.05). This finding is consistent with a study demonstrating that alteration in BRCA1 gene expression significantly correlates with higher breast cancer histological grade (Lee, 2002). Grade is an independent prognostic indicator and is inversely related to outcome (Elston and Ellis, 1991). Several reports have indicated that a specific histopathological phenotype can be recognized in breast carcinoma occurring in women with germline mutations in BRCA1 (Elston and Ellis, 1991; Stratton, 1997). One of these studies reported an increased rate of ductal carcinoma in breast cancers associated with BRCA1 mutations (Elston and Ellis, 1991). Others reported an increase in the incidence of tumors with a medullary and atypical medullary pattern within the group of ductal carcinomas associated with BRCA1 mutations (Marcus et al., 1996; Stratton, 1997). The findings of the present study support the findings of these workers. However, 40% of breast cancers with alteration in BRCA1 mRNA in the study were mainly high-grade carcinoma. Moreover, this study showed an association between BRCA1 expression and negative ER and PR expression (statistically significant). Previous reports also showed that breast cancers associated with BRCA1 mutation were significantly more often ER and PR negative (Marcus et al., 1996; Lakhani et al., 2002). ER has become one of the most important prognostic and predictive markers for breast cancer (Lakhani et al., 2002). ER-positive tumors tend to grow more slowly, are better differentiated, and are associated with slightly better overall prognosis (Karp et al., 1997). PR status is also a good predictor of tumor responsiveness to therapy. Approximately 75% of ER/PR-positive tumors respond positively to endocrine therapy (Clark, 2000; Elledge and Fuqua, 2000). In addition to the above relationship with hormone receptors, the present study also demonstrated a positive relationship between overexpression of the protooncogene HER-2 in breast cancers with alteration of BRCA1 mRNA (33% of cases), one of which was CK5/6 positive. Our findings are in agreement with the study by Yoshikawa et al. (1999), who reported that breast cancers with reduced BRCA1 protein expression had a tendency toward overexpression of HER-2. This protooncogene is a biological marker for breast cancer, overexpression of which indicates poor prognosis. Furthermore, HER-2 is amplified in ∼20% of invasive cancers and has received interest because of its association with lymph node metastasis and its short relapse time, poor survival, and decreased response to endocrine and chemotherapy (Varley et al., 1987). Antibodies directed against the HER-2 protein have attracted a lot of attention because of the availability of the monoclonal antibody Herceptin for treatment of breast cancer (Slamon et al., 2001). Some studies have reported that breast cancers associated with BRCA1 mutations develop at a relatively early age (Elston and Ellis, 1991). Women carrying a BRCA1 or BRCA2 mutation are known to have an increased risk of developing contralateral primary breast cancer (Metcalfe et al., 2004), which is even more apparent among younger (age <50 years) women diagnosed with a primary breast carcinoma (Robson et al., 1998; Verhoog et al., 2000). In another study, a BRCA1 mutation carrier presented with metastatic disease 3.5 years after prophylactic mastectomy (no primary breast cancer found), suggesting the presence of an occult primary tumor that was never found, despite a thorough reexamination of the specimen at the time of presentation with the metastatic disease. This finding emphasizes the fact that, despite thorough examination of the mastectomy specimens, the presence of an occult breast cancer cannot be ruled out completely and indicates that a form of surveillance after prophylactic mastectomy might be relevant (Meijers-Heijboer et al., 2001). BRCA1 or BRCA2 mutation carriers and women from a hereditary breast/ovarian cancer family have a highly increased risk of developing breast cancer. Prophylactic mastectomy results in the greatest reduction in breast cancer risk (Heemskerk-Gerritsen et al., 2007).
BRCA1 has a distinctive morphology and immunohistochemical phenotype. Combined morphological and immunohistochemical data can be used to predict the risk in a young Egyptian patient harboring a germline mutation in BRCA1.
Conflicts of interest
There are no conflicts of interest.
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